The expression of CD154 (CD40 ligand) by activated T lymphocytes is critical in the development of humoral and cell-mediated immunity (Foy, et. al. (1996) Annu. Rev. Immunol. 14:591–617; Grewal and Flavell (1998) Ann. Rev. Immunol. 16:111–135; Noelle (1996) Immunity 4:415–419). The interaction of CD154 with its receptor, CD40, was first shown essential for B cell growth and differentiation and formation of germinal centers (Foy, et. al. (1996) Annu. Rev. Immunol. 14:591–617). This interaction is essential to numerous elements of cell-mediated immunity. In the absence of CD154, antigen presentation by dendritic cells and macrophages is profoundly impaired, as is macrophage-mediated killing of intracellular or extracellular pathogens (Grewal and Flavell (1998) supra; Noelle (1996) supra). Given the breadth of importance of CD154-CD40 interaction, it is not surprising that CD154 blockade retards the development and progression of immune responses in an array of transplantation and autoimmune disease models ranging from Systemic Lupus Erythematosus to Rheumatoid Arthritis to Multiple Sclerosis (Foy, et. al. (1996) supra; Grewal and Flavell (1998) supra).
The CD154 gene is located on the X chromosome, and belongs to the Tumor Necrosis Factor (TNF) gene family (Hollenbaugh, et al. (1994) Immunol. Rev. 138:23–37). Study of CD154 expression chiefly involves CD4+ T lymphocytes, with the earliest studies showing that resting cells express little or no CD154 (Lane, et al. (1992) Eur. J. Immunol. 22:2573–2578; Nusslein, et al. (1996) Eur. J. Immunol. 26:846–850; Roy, et al. (1993) J. Immunol. 151:2497–2510). Activation of the T lymphocytes demonstrated that induction of CD154 expression was different from that of other cytokines. Signals (anti-CD3, mitogenic lectins) that triggered resting T cells to engage in high levels of proliferation and cytokine production would elicit very little (CD4+ T cells) or no (CD8+ T cells) expression on either mouse or human T cells (Lane, et al. (1992) supra; Nusslein, et al. (1996) supra; Roy, et al. (1993) supra). Optimal expression of CD154 was found to require pharmacologic stimulation provided by phorbol myristate acetate (PMA) and calcium ionophores such as ionomycin (Lane, et al. (1992) supra; Nusslein, et al. (1996) supra; Roy, et al. (1993) supra; Roy, et al. (1994) Eur. J. Immunol. 25:596–603). The induction of CD154 on T lymphocytes is blocked by concurrent treatment with cyclosporine and glucocorticoids; these effects are presumed to be transcriptional (Fuleihan, et al. (1994) J. Clin. Invest. 93:1315–1320; Roy, et al. (1993) supra) based on the presence of NF-AT sites in the CD154 promoter (Schubert, et al. (1995) J. Biol. Chem. 15:29264–29627). Since cyclosporine and glucocorticoids also inhibit cytokine production (Ashwell, et al. (1992) Ann. Rev. Immunol. 18:309–345; Sigal and Dumont (1992) Ann. Rev. Immunol. 10:519–60), this pathway does not account for the differential regulation of CD154 expression by T lymphocytes.
The expression of TNF-α is primarily regulated at the level of mRNA turnover and translation, conferred by adenine-uridine rich cis-acting elements (AURE) present in its 3′-untranslated region (Beutler and Kruys (1995) J. Cardiovasc. Pharm. 25:S1–8; Kontoyiannis, et al. (1999) Immunity 10:387–398; Shaw and Kamen (1986) Cell 46:659–669). CD154 mRNA is rapidly degraded in human peripheral blood T lymphocytes, with a half-life of approximately 30 minutes, similar to that of interleukin 2 (Ford, et al. (1999) J. Immunol. 162:4037–4044; Murakami, et al. (1999) J. Immunol. 163:2667–2673; Rigby, et al. (1999) J. Immunol. 163:4199–4206; Suarez, et al. (1997) Eur. J. Immunol. 27:2822–2829). CD154 and cytokine mRNA stability may be differentially regulated in activated T lymphocytes as evidenced by CD2 engagement by LFA-3 stabilizes CD154 mRNA without altering IL-2 mRNA stability (Murakami, et al. (1999) supra) and CD28 crosslinking increases cytokine (TNF-α, IL-2) production at the level of mRNA stability (Lindsten, et al. (1989) Science 244:339–343) while having minimal effect on CD154 expression (Ford, et al. (1999) supra).
Using human peripheral blood lymphocytes (PBL) it was observed that PMA or ionomycin treatment rapidly increased CD154 mRNA stability, even in the context of transcriptional inhibition (Rigby, et al. (1999) supra). In these studies, two major, p50 and p25, and two minor, p40 and p36, RNA binding proteins were shown to bind the CD154 3′-untranslated region. The binding of the p50 and p25 mapped to a polypyrimidine-rich region (˜0.4 kb) that lacked an AURE. UV crosslinking studies demonstrated that the p50 and p25 directly contacted uridines and cytidines in this region. Signals which stabilized CD154 mRNA decreased p25 levels in both cytosolic and polysomal fractions, while a corresponding increase in p50 binding activity was observed (Rigby, et al. (1999) supra).
It has now been found that a novel cis-acting element in this polypyrimidine-rich region exists and regulates CD154 mRNA turnover through the relative levels of two polypyrimidine tract binding proteins.